The past decade has witnessed the rapid emergence of genetic engineering technology coupled with major improvements in the development of large-scale insect cell culture processes. These developments have led to the widespread use of insect cells as a valuable host cell system for the expression of recombinant proteins.
One of the insect cell lines which has shown great promise is BTI-Tn-5B1-4, established at Boyce Thompson Institute, Ithaca, N.Y. and commercially available as High Five.TM. cells from Invitrogen Corp. The cell line is on deposit at the American Type Culture Collection as ATCC CRL 10859. This novel cell line is the subject of copending patent application Ser. No. 07/983,821, filed Dec. 1, 1992. These cells were derived from eggs of the Cabbage Looper (Trichoplusia ni). They have been found to be particularly susceptible to baculoviruses, which are adaptable to genetic modifications which lead to high levels of secretion of proteins.
Since this time, the BTI-Tn-5B1-4 cells have been maintained continuously as an anchorage dependent cell line, first in complete medium, and now in both complete and serum-free medium. By "anchorage dependent", we mean that the cell line forms layers and clumps attached to the vessel in which the cells are cultivated. The above-referenced copending application is directed to these cells in an anchorage-dependent form, as no means had been found at that time to adapt the cell line to grow in suspension.
Several research groups, inside and outside Boyce Thompson Institute, the assignee of this application and the above-referenced copending applications, have attempted to adapt this cell line to suspension conditions, i.e. maintenance of the cell line as individual cells suspended in a medium. All have met with the same result: severe clumping.
The anchorage dependency of the cells presents no problem for research, but limits the applicability of the cell line for commercial applications where in order to scale up the production it is desirable to maintain the cell lines in bioreactors (i.e., in suspension).
It will be understood by those skilled in the art that the problem of clumping or anchorage-dependency is not limited to the BTI-Tn-5B1-4 cell line, but is common to many cell lines.
Heparin is "an acid mucopolysaccharide acting as an antithrombin, antithromboplastin, and antiplatelet factor to prolong the clotting time of whole blood. It occurs in a variety of tissues, most abundantly in liver." (McGraw-Hill Dictionary of Scientific and Technical Terms, 1978, p.741)
Heparin acts as an anticoagulant by increasing the rate of formation of irreversible complexes between antithrombin III and the serine protease clotting factors. (Lubert Stryer, Biochemistry, 1988, p.255)
Although heparin is a known anticoagulant for blood cells, as shown by the above references, its effect upon cell lines is less clear. It will be noted that the following references all deal with human or mammalian cell lines, or with blood. We are aware of no prior art which deals with the effect of heparin upon insect cells.
Carroll, Ippolito, and DeWolf (Div. Cell. Genet., Charles A. Dana Res. Inst., Beth Israel Hosp., Boston) reported that human teratocarcinoma (HT) cell line HT-D (1218E) formed 40-60% rosettes which was inhibited by addition of heparin to the cell suspensions. . . . Heparin was capable of disrupting monolayer formation by cell line HT-D (1218E) when added to cultured cells growing in vitro. These results suggest that a heparin, or more likely, heparan sulfate-binding component on the surface of some human teratocarcinoma cell lines may be involved in cellular adhesion."--HEPARIN-BINDING AGGLUTININ ON HUMAN TERATOCARCINOMA CELLS; BIOCHEM. BIOPHYS. RES. COMMUN., 1982, 109/4 (1353-1359)
Note: "Heparan Sulfate is like heparin except it has fewer N- and O-Sulfate groups and more N-acetyl groups", Stryer Biochemistry, 1988, p 276. For the purposes of this disclosure, heparin and heparan sulfate are considered equivalent and will be referred to generically as "heparin".
The above reference would seem to teach that heparin inhibits aggregation. However, the following references would indicate exactly the opposite: that heparin induces aggregation of cells.
Thurn and Underhill (Department of Anatomy, School of Medicine and Dentistry, Georgetown University, Washington, D.C.) reported that "The effects of different carbohydrates on cell-to-cell adhesion were examined in an aggregation assay, which consisted of swirling a suspension of cells and monitoring the loss of single cells with a Coulter Counter. Of the carbohydrates tested, only heparin and dextran sulfate induced cell aggregation. This effect occurred in freshly isolated mouse splenocytes and in cultured cells of lymphoid origin (P388, YAA-Cl) but not in cell lines of fibroblastic origins (3T3, SV-3T3, BHK, and PY-BHK). Using the YAA-Cl cell line for further study, we found that aggregation could be induced by relatively small amounts of heparin (&lt;10 .mu.g/ml)." (emphasis added)--HEPARIN-INDUCED AGGREGATION OF LYMPHOID CELLS; J. CELL. PHYSIOL., 1986, 126/3 (352-358)
Gill, Silbert and Silbert (Department of Medicine, Harvard Medical School, Boston, Mass.) reported the following experiments: "Human skin fibroblasts and calf aorta endothelial cells were grown as tissue culture monolayers in the presence of (sup 3sup 5S) sulfate in order to label the glycosaminoglycan portions of proteoglycans for investigation of their role in cell attachment. The (sup 3sup 5S) glycosaminoglycans were then selectively removed from the cell monolayers by the addition of various glycosaminoglycan-degrading enzymes. As previously described, in contrast to trypsin treatment none of these enzymes removed any cells from the culture plates. Incubation with a preparation from Flavobacterium heparinum left only small stubs of (sup 3sup 5S) glycosaminoglycans on the cell monolayers, indicating that all the cell-surface proteoheparan (sup 3sup 5S) sulfate and proteochondroitin (sup 3sup 5S) sulfate was accessible to this enzyme preparation. The treatment did not change the amount or time of incubation with trypsin necessary for release of the cells from the monolayers. Thus, cell attachment was not weakened by removal of heparan sulfate or chondroitin sulfate. In contrast, neither fibroblasts nor endothelial cells in suspension would reattach in the presence of the F. heparinum preparation while reattachment occurred readily in the presence of chondroitin ABC lyase. This provides evidence that heparan sulfate, but not chondroitin sulfate, is involved in the process of cell attachment even though neither is necessary for maintaining attachment."--EFFECTS OF HEPARAN SULFATE REMOVAL ON ATTACHMENT AND REATTACHMENT OF FIBROBLASTS AND ENDOTHELIAL CELLS;
BIOCHEMISTRY, 1986, 25/2 (405-410)
See also: Moore, Mahaffey, and Zboran (Dep. Of Large Animal Med. And Physiol.-pharmacol., Coil. Of Vet. Med., Univ. Of Ga., Athens, Ga.), HEPARIN-INDUCED AGGLUTINATION OF ERYTHROCYTES IN HORSES; American Journal of Veterinary Research 48 (1). 1987; and Mahaffey and Moore (Dep. Vet. Path., Coll. Vet. Med., Univ., Athens, Ga.), ERYTHROCYTE AGGLUTINATION ASSOCIATED WITH HEPARIN TREATMENT IN THREE HORSES J. American Veterinary Medical Association 1986. 189 (11):1478-1480 (10 ref.) (Note: "Erythrocytes" are also known as "red blood cells").
Since the primary reason for experimentation with the insect cell lines which led to the invention is the production of various substances through genetic engineering, it was considered important that heparin not interfere with this process. The following reference would appear to teach away from the use of heparin in this application:
"In experiments on CBA mice in which a 2% suspension of red blood cells was used as the antigen, heparin substantially prevented the migration of antibodies from antibody-producing cells in vitro. . . . It is concluded that heparin is able to interact with the outer membrane of immunocompetent cells and to inhibit migration of antibodies synthesized by them into the surrounding medium."--Kozlov, Kaznacheev, Lozovoi, and Lyakhovich (Lab. Clin. Immunopathol., Inst. Clin. Exp. Med., Siberian Branch, Acad. Med. Sci. USSR), MECHANISM OF ACTION OF HEPARIN OF LYMPHOCYTES IN VITRO; BULL. EXP. BIOL. MED., 1978, 84/11 (1612-1614)
Dougherty, et. al. discussed the effect of heparin, among other substances, on enhancing virus absorption by insect cell lines in an article entitled "Physical Factors That Affect In Vitro Autographica californica Nuclear Polyhedrosis Virus Infection" (Applied and Environmental Microbiology, May 1981, pp. 1166-1172). The reference does not discuss the effect of heparin on cell propagation or attachment. The cells were not passaged serially in the presence of heparin, nor was selection for non-attachment dependent cells performed.